The present invention relates to the field of three-dimensional matrices that contain pharmacologically active molecules, particularly growth factors. The invention also relates to the use of growth factors or proteins in a matrix designed to promote cell and tissue growth. The invention further relates to the use of growth factors with low heparin-binding affinity. In addition, the invention relates to the field of articles of manufacture useful as implantable devices and wound dressings as the matrix of the invention is designed to be used in conjunction with such devices to provide protracted and controlled release of growth factor, thus promoting wound healing in the patient.
Many growth factors are thought of as xe2x80x9cheparin-bindingxe2x80x9d growth factors. Families with one or more members that bind heparin include fibroblast growth factors and bone morphogenetic proteins (BMPs) (1, 2). Additional growth factors that bind heparin include transforming growth factor xcex21 (TGF-xcex21), interleukin-8, neurotrophin-6, vascular endothelial cell growth factor, heparin-binding epidermal growth factor, hepatocyte growth factor, connective tissue growth factor, midkine, and heparin-binding growth associate molecule (3-11). These factors have shown the potential to enhance healing in many different types of tissue including vasculature, skin, nerve, and liver.
Controlled delivery devices based on heparin-affinity of these growth factors have been designed previously (12-14). These drug delivery devices have previously been used to deliver xe2x80x9cheparin-bindingxe2x80x9d growth factors. Such xe2x80x9cheparin-bindingxe2x80x9d growth factors are typically considered to be those which bind to heparin with a relatively high affinity, often characterized by elution from heparin-affinity columns at NaCl concentrations well above physiological levels ( greater than 140 mM). In such delivery systems, the heparin-binding affinity of the growth factor is usually used to sequester the growth factors to immobilized heparin of some form. For example, Edelman et al. have used heparin-conjugated Sepharose beads to bind basic fibroblast growth factor (bFGF) and then encapsulated the beads with alginate (12, 19). These beads serve as reservoirs that release bFGF slowly based on the binding and dissociation constants of bFGF and heparin.
The delivery of xe2x80x9cnon-heparin-binding growth factorsxe2x80x9d has previously required release methods for delivery typically based on diffusion-controlled release of the factors from porous materials (15-18). There remains a need in the medical arts for a device that is capable of providing the release of low heparin-binding growth factors at a controlled and predictable rate in order-to provide effective release of the factor over a clinically useful period during the wound healing process.
In a general and overall sense, the present invention relates to the use of how non-heparin-binding growth factors in delivery techniques employing growth factors with heparin-affinity by utilizing low heparin affinity sequences present in many proteins. The particular growth factors employed as part of the invention have been found by the present inventors to possess a basic sequence at a site in the protein that is freely accessible in the proteins native conformation. This basic region may possess only relatively low heparin-affinity.
As used in the description of the present invention, xe2x80x9clow-heparin-binding affinityxe2x80x9d of a growth factor or peptide fragment thereof is defined as any protein, peptide, or derivative or combination thereof, that is capable of demonstrating the biological activity of a growth factor, and that has a relatively binding low affinity for binding heparin, and will elute from a heparin-affinity column at sub-physiological NaCl concentrations. Physiological levels of NaCl may be defined as about 140 mM NaCl. Herein the term xe2x80x9csub-physiologicalxe2x80x9d levels of NaCl, therefore, may be further defined as from between about 25 mM to about 140 mM NaCl. Although low heparin-binding affinity growth factors elute from heparin-affinity columns at sub physiological NcCl concentrations, their low affinity for heparin can still be used to sequester the protein or peptide to a matrix that contains heparin or a heparin-binding site.
By way of example, and in no way intending to be limited to any particular mechanism of action, the invention may be described as employing a matrix having growth factor proteins with a relatively high-ratio of heparin-binding sites. A ratio of at least 1:1 heparin to growth factor must be used, but the greater the excess of heparin sites the slower the release. In this fashion, primarily xe2x80x9cnon-heparin-bindingxe2x80x9d growth factors or peptide fragments thereof with relatively low heparin-binding affinity can be bound to a heparin-decorated matrix. These matrices can then serve as reservoirs containing the growth factor or factors to be delivered. The dissociation kinetics of low affinity heparin-binding proteins are relatively fast, but the high number of binding sites allows rebinding of the growth factor before it can diffuse out of the matrix. Release can occur by diffusion of the growth factor out of the matrix prior to rebinding, or it can occur if the growth factor encounters a cell surface receptor before rebinding to a heparin site. In this fashion, release of the growth factor or bioactive fragment thereof can be sustained, and continue to foster improved healing.
In a general and overall sense, the present invention describes in at least one aspect a specially designed matrix that provides for the release of growth factors or bioactive fragments thereof. The growth factor is defined as having low binding affinity for heparin. The matrix, more particularly, may be defined as comprising a substrate capable of providing attachment of heparin, a heparin-like polysaccharide, or a heparin-like polymer, and a growth factor or peptide fragment thereof having a basic domain that binds heparin with low affinity.
The characteristic of the growth factor or peptide fragment thereof as binding to heparin with low affinity may be further described as a peptide/protein that will elute from a heparin affinity column at an NaCl concentration of about 25 mM to about 140 mM.
The xe2x80x9clow heparin-binding affinityxe2x80x9d growth factor or peptide fragment thereof may be further defined as comprising a length of about 8 to 30 amino acid residues. This sequence of amino acid residues, in some embodiments, may be defined as comprising at least 2 basic amino acid residues, a ratio of basic to acidic amino acid residues of at least 2, and a ratio of hydrophobic amino acid residues to basic amino acid residue of at least 0.67. The growth factor or fragment thereof elutes from a heparin affinity column at less than 140 mM or at about 25 to aboutl 40 mM NaCl.
For purposes of this application, basic amino acids may be defined as K (lysine) or R (arginine). The acidic amino acid residues may be further defined as D (aspartic acid) or E (glutamic acid). The hydrophobic amino acid residues may be defined as A (alanine), V (valine), F (phenylalanine), P (proline), M (methionine), I (isoleucine), or L (leucine). For purposes of this application, C (cysteine) that are involved in a disulfide bridge are also considered hydrophobic.
By way of example, the low heparin-binding affinity growth factor or a peptide fragment thereof as defined in the invention comprises neurturin, persephin, IGF-1A, IGF-1xcex2, EGF, NGFxcex2, NT-3, BDNF, NT-4, TGF-xcex22, TGF-xcex23, TGF-xcex24, or a peptide fragment of any one of these. Other growth factors may be found which contain similar basic domains that are not enumerated here. The matrix itself may also comprise any of a variety of materials, such as fibrin, collagen, hyaluronic acid, or a synthetic polymer hydrogel. By way of example, the synthetic polymer hydrogel may be a poly (ethylene glycol) hydrogel or a derivative thereof. Other synthetic polymer hydrogels may be used apart from those enumerated here.
The peptides of the invention that bind heparin with high affinity have a characteristic amino acid domain that will not elute from a heparin-affinity column at less than 140 mM NaCl. While many potential peptides exist, the inventors have identified several peptide sequences in particular. These are exemplified in the amino acid sequences identified in SEQ. ID. NO.:1, SEQ ID. NO.:2; SEQ ID. NO.:3; SEQ ID. NO.:4; and SEQ ID. NO.:5. Many other peptides may be used apart from the specifically enumerated sequences here.
The heparin or heparin-like polysaccharides of the invention may be further characterized, at least in some embodiments, as having a molecular weight of at least 3,000 Daltons. It is contemplated that virtually any molecular weight heparin or heparin-like polysaccharide could be used in the practice of the invention of at least 3,000 Daltons, without any upper molecular weight limitation. For practical purposes a molecular weight maximum of 10,000,000 may be considered. In particular applications, the heparin-like polysaccharide may be further defined as a polysaccharide having a molecular weight of at least 3,000 Daltons and having at least one negative charge per two saccharide rings and no more than one positive charge per ten saccharide rings.
Examples of heparin-like polysaccharide include dextran sulfate, chondroitin sulfate, heparin sulfate, fucan, alginate or derivatives thereof. The present inventors have found that particular preparations of the matrix that include a particular molar ratio of heparin to growth factor, such as a molar ratio of 1, may be employed in the practice of the invention. It has further been found that a matrix of the invention that includes a molar ratio of covalently-attached peptide having a binding domain that binds heparin with high affinity to heparin or a heparin-like polysaccharide of at least one is in some embodiments of the matrix a preferred ratio. These heparin and heparin-like polysaccharides may be either covalently attached to the substrate or immobilized via non-covalent interactions (i.e. electrostatically bound). Synthetic polymers may be designed that function in a heparin-like manner.
The substrate of the matrix as defined in the present invention may comprise fibrin, collagen, hyaluronic acid, a synthetic polymer gel, a mixture thereof, or any variety of synthetic derivatives thereof, that is capable of supporting the attachment of the types of peptides described there, and/or cells.
The matrix and various embodiments of the matrix described herein provide a multitude of advantages, particularly when employed at tissue sites where a wound healing response is desired. The growth factor or peptide fragment thereof provided in the matrix is released by the degradation of a component of the matrix, by the disassociation of growth factor from the heparin or heparin-like polysaccharide, or by a combination of these mechanisms. In this manner, a more sustained and controlled, release of growth factor into the site where the matrix is implanted may be achieved. Methods for providing the controlled release of growth factor at a wound site in need thereof are provided by the present invention. The growth factor for such applications may include TGF-xcex23, which may be particularly useful in dermal healing. The invention provides various articles of manufacture, such as a vascular graft or shunt, or tissue replacement that includes the matrix defined in this disclosure. Such articles may in some embodiments be defined as implantable sterilized compositions. The heparin-binding peptide used in the present examples possesses high heparin affinity.
In addition to using protein matrices as the substrate of the delivery system, other types of matrices can be used. Synthetic polymer hydrogels, including hydrogels formed by photopolymerization or conjugate addition reactions, can be utilized as the substrate for the delivery device. This synthetic material may contain cell adhesion domains, substrates for enzymatic degradation or hydrolysis, heparin-binding domains, or covalently bound heparin. Through either the covalent or non-covalent attachment of heparin, such synthetic matrices can bind low heparin-binding affinity growth factor proteins and release them in a controlled manner. Release can occur by degradation of matrix components or dissociation of the low heparin-binding affinity growth factor proteins, just as in protein matrices.
The substrate for the delivery system can also include matrices of hyaluronic acid or hyaluronic acid derivatives. Such materials are commonly used and readily available. The addition of covalently or non-covalently bound heparin or heparin-like polysaccharides can be used to provide controlled delivery of low heparin-binding affinity growth factor proteins. Release can occur by degradation of matrix components or dissociation of the low heparin-binding affinity growth factor proteins, just as in protein or synthetic polymer matrices.
In addition to heparin, other heparin-like polymers have similar binding affinity for heparin-binding proteins and peptides. Such heparin-like polymers include, for example, dextran sulfate, chondroitin sulfate, heparin sulfate, fucan and alginate (See Maaroufi, et al, 1997 (46) and Logeart, et al., (1997) (47). In addition, synthetic heparin-like polymers or polysaccharide derivatives also exist, which have similar binding affinity for heparin-binding proteins or peptides as heparin. Examples of heparin-like polysaccharide derivatives include dextran derivatives such as those made by de Raucourt, et al., (1998) (48) and Bagheri-Yamand, et al., (1998) (49). Examples of heparin-like synthetic polymers include those by Silver, et al., (1992) (50). For the purposes of this invention, usage of the term xe2x80x9cheparinxe2x80x9d is considered to include all heparin-like polymers and polysaccharides including those described above.
(1) Amino acid three-letter and one-letter abbreviations:
The following sequences are referenced in the description of the present invention.